Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Synthesis of substrates
2,2-[2H2]-Decanoyl-CoA (3) was synthesised using an extension of the method previously
described by us.1 Thus, diethyl malonate 7 (Scheme 1) was deprotonated and the resulting anion
alkylated with 1-bromooctane to give the mono-substituted malonate 8. Prolonged treatment with
boiling concentrated 2HCl in 2H2O exchanged the α-proton with deuterium, hydrolysed the ethyl
esters and caused decarboxylation of the intermediate 2-2H-2-octylmalonic acid to give the required
di-deuterated decanoic acid 9. This was then coupled with CoA using ethyl chloroformate by our
previously reported method1 to provide the corresponding CoA ester 3.
The required 13C-decanoyl-CoA substrate 4 was synthesised by analogy to the method for the
dideuterio compound 3 above. Thus, reaction of the anion derived from diethyl 2-[13C]-malonate 10
with 1-bromooctane gave 11. Hydrolysis of the esters and decarboxylation with boiling aq. 1HCl,
furnished the labeled acid 12 in 54% overall yield. Coupling with CoA gave the desired substrate 4
(Scheme 1).
Scheme 1. Syntheses of 2,2-[2H2]-decanoyl CoA 3 and of 2-[13C]-decanoyl CoA 4. Reagents and
yields: i, NaH, 1-bromooctane, DMF, 52% (8), 59% (11)1; ii, 35% 2HCl / 2H2O, reflux, 8 d, 51%;
iii, EtOCOCl, Et3N, CoASH1; iv, 35% 1HCl / 1H2O, reflux, 90%.
Stereochemical analysis of reaction products
Decanoyl-CoA 2 was incubated with active enzyme until ca. 60% exchange of the α-protons with
deuterium had occurred. Following quenching of the reaction, the CoA ester was hydrolysed under
alkaline conditions. The reaction mixture was then acidified and the decanoic acid isotopomeric
mixture 13 was extracted into dichloromethane. The carboxylic acids were converted into their acid
chlorides by treatment with oxalyl chloride; these acid chlorides were then used to acylate the free
OH group of R-2-mandelate methyl ester to form the diastereomeric decanoyl esters 6.
Scheme 2. Synthesis and structure of methyl O-decanoyl-R-mandelate 6. Reagents and yields: i,
(COCl)2, DMF, CH2Cl2; ii, methyl R-mandelate, Et3N, DMAP, CH2Cl2, 81% over 2 steps.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Experimental
General: All chemicals were obtained from the Sigma-Aldrich Chemical Co. or Fisher Ltd. and
were used without further purification, unless otherwise specified. Experiments were conducted at
ambient temperature, unless noted otherwise. Organic extracts were dried over MgSO4 and filtered.
Solvents were evaporated under reduced pressure. Melting points are uncorrected. NMR spectra
were obtained on JEOL Eclipse Delta (270 MHz), Varian Mercury (400 MHz) or Varian Inova (600
MHz) spectrometers, in CDCl3 solution, except where noted. Coupling constants (J) are reported in
Hz to the nearest 0.5 Hz. Decanoyl-CoA, octanoyl-CoA, butanoyl-CoA and acetyl-CoA were
purchased as their Li3 salts from Larodan Fine Chemicals AB, Malmö, Sweden. 2-
Methylpropanoyl-CoA, pentanoyl-CoA, hexanoyl-CoA, heptanoyl-CoA, labeled decanoyl-CoA and
S-2-methyldecanoyl-CoA esters were synthesised from their corresponding acids by the reported
method.1 Recombinant human AMACR 1A was purified as described.1
Ethyl 2-ethoxycarbonyldecanoate (8). Diethyl malonate 7 (2.00 g, 12.5 mmol) was treated with
NaH (275 mg, 6.87 mmol) and 1-bromooctane (1.20 g, 6.25 mmol) in dry DMF (50 mL), as
previously described for the 2-[13C] isotopomer,1 to give 8 (890 mg, 52%) as a colourless oil: δH
0.72 (3 H, t, J = 7.0, 10-CH3), 1.00-1.25 (18 H, m, 6 × CH2 + 2 × CH3), 1.76 (2 H, m, 3-CH2), 3.17
(1 H, t, J = 7.4, 2-CH); 4.06 (4 H, q, J = 7.0, 2 × OCH2).
2-[2H2]-Decanoic acid (9). Diester 8 (890 mg, 3.27 mmol) was treated with 35% 2HCl / 2H2O (10
mL) at reflux for 8 d. Work-up as for the 2-[2H2]-2-[13C] isotopomer1 gave 9 (289 mg, 51%) as a
colourless oil: δH 0.86 (3 H, t, J = 6.6, 10-CH3), 1.20-1.40 (12 H, m, 6 × CH2), 1.60 (2 H, m, 3-H2);
MS (ES-) m/z 174.1544 (M – H) (13C112C9
2H21H17
16O2 requires 174.1545), 173.1510 (100%; M – H)
(12C102H2
1H1716O2 requires 173.1511).
2-[13C]-Decanoic acid (12). Ethyl 2-[13C]-2-ethoxycarbonyldecanoate 111 (1.00 g, 3.7 mmol) was
treated with aq. HCl, as for the synthesis of the 2-[2H2]-2-[13C] isotopomer,1 to give 12 (570 mg,
90%) as a colourless oil: 1H NMR δH 0.87 (3 H, t, J 6.8, 10-CH3), 1.27 (12 H, m, 6 × CH2), 1.60 (2
H, m, 3-H2), 2.40 (2 H, dt, J = 135.2, 7.4 Hz, 2-13CH2); 13C NMR δC 14.08, 22.64, 24.66 (d, J =
33.8, 3-C), 29.04 (4-C), 29.25 (d, J = 33.8, 5-C), 29.37 (C-6), 31.84, 33.89 (d, J = 55.2, 2-C for 1,2-13C2 isotopomer), 33.90 (enhanced, 2-C), 33.90 (d, J = 34.5, 2-C for 2,3-13C2 isotopomer), 179.28
(d, J = 55.2, 1-C); MS (ES-) m/z 174.1476 (0.7%; M – H) (12C713C3H19O2 requires 174.1486),
173.1453 (8.8%; M – H) (12C813C2H19O2 requires 173.1453), 172.1419 (100%; M – H)
(12C913C1H19O2 requires 172.1419).
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Methyl R-2-(decanoyloxy)phenylacetate (6). Decanoic acid (400 mg, 2.32 mmol) was treated with
oxalyl chloride (590 mg, 4.64 mmol) and DMF (20 μL) in CH2Cl2 (5.0 mL). The evaporation
residue, in CH2Cl2 (5.0 mL) was stirred with methyl R-2-hydroxyphenylacetate (385 mg, 2.32
mmol), Et3N (470 mg, 4.64 mmol) and 4-dimethylaminopyridine (1.0 mg) under N2 for 16 h.
Washing (aq. citric acid, brine), drying (MgSO4) and evaporation gave 6 (625 mg, 81%) as a pale
yellow oil: δH 0.85 (3 H, t, J = 6.8, decanoyl 10-CH3), 1.23-1.34 (12 H, m, 6 × CH2), 1.63-1.68 (2
H, m, decanoyl 3-H2); 2.38-2.47 (2 H, m, decanoyl 2-H2); 3.70 (3 H, s, OMe); 5.90 (1 H, s,
CHCO2Me); 7.23-7.45 (5 H, m, Ph-H5). Irradiation at δ 1.65 resolved the peaks at δ 2.28-2.37 into
two doublets at δ 2.30 and δ 2.37, with 2J = 15 Hz.
Enzyme assays: Assays were conducted in a final volume of 0.7 mL in buffer as previously
described,1 using purified recombinant AMACR 1A (54 μg, 1.15 nmol). Incubations were at 30°C
and the reactions were terminated at the required time by heating at 50°C for 10 min. Incubations
of acyl-CoAs used the following substrates (Fig. 3 of main paper): decanoyl-CoA 2, octanoyl-CoA,
heptanoyl-CoA, hexanoyl-CoA, pentanoyl-CoA, butanoyl-CoA, acetyl-CoA and 2-
methylpropanoyl-CoA were performed in buffer containing 2H2O and were analysed directly by 1H
NMR at 400 or 600 MHz. Incubations of 2-[13C]-decanoyl-CoA 4 were performed in buffer
containing 2H2O and were analysed directly by 13C NMR at 100.6 MHz. Incubations of 2,2’-[2H2]-
decanoyl-CoA 3 were performed in buffer containing 1H2O and were analysed by 1H NMR; 2H2O to
ca. 10% (v/v) was added following termination of the reaction before analysis. All data is based on
two replicants of each substrate concentration and substrate conversions were corrected for non-
enzymatic exchange levels in negative controls. Concentrations of acyl-CoA esters were determined
by UV-visible spectroscopy, using ε260 = 16 mM-1 cm-1.2
Kinetic analyses used six different concentrations of substrate which were incubated for 1 h (S-2-
methyldecanoyl-CoA 1S) or 4 h (decanoyl-CoA 2). Data was corrected for non-enzymatic exchange
levels in negative controls, and analysed using SigmaPlot 10.0.1 with enzyme kinetics module 1.3
and parameters estimated using the direct linear plot.3, 4 The kinetic parameter kcat was calculated
assuming a molecular mass of 47146.8 Da for AMACR.1 Parameters are also calculated using the
Michaelis-Menten plot and are report ± standard error.
Determination of configuration of deuterated enzymatic products. Decanoyl-CoA 2 was
exchanged using AMACR to ca. 60% conversion as described above. The products were
hydrolysed (NaOH) and acidified1 and the exchanged acids 13 were extracted (CH2Cl2) and dried
(MgSO4). The evaporation residue (ca. 500 μg, 2.90 μmol) was dissolved in CH2Cl2 (25 μL) and
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
treated with oxalyl chloride (2.45 μL, 29 μmol) and DMF (0.5 μL) for 6 h under N2. The
evaporation residue, in CDCl3 (90 μL), was treated with 4-dimethylaminopyridine (DMAP) (90 μg,
700 nmol), methyl R-2-hydroxyphenylacetate (methyl R-mandelate) (480 μg, 2.90 μmol) and Et3N
(800 nL, 5.8 μmol) under N2 for 16 h. The mixture was washed (aq. citric acid), dried (MgSO4) and
diluted to 700 μL with CDCl3 for 600 MHz NMR analysis.5 The signal at δ 1.65 (3-CH2) was
irradiated during NMR analysis to decouple and simplify the signals in the α-CH2 region.
References
1. D. Darley, D. S. Butler, S. J. Prideaux, T. W. Thornton, A. D. Wilson, T. J. Woodman, M.
D. Threadgill and M. D. Lloyd, Org. Biomol. Chem., 2009, 7, 543-552.
2. I. Rudik and C. Thorpe, Arch. Biochem. Biophys., 2001, 392, 341-348.
3. A. Cornish-Bowden and R. Eisenthal, Biochem. J., 1974, 139, 721-730.
4. R. Eisenthal and A. Cornish-Bowden, Biochem. J., 1974, 139, 715-720.
5. D. Parker, J. Chem. Soc.-Perkin Trans. 2, 1983, 83-88.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Enzyme kinetic data for S-2-methyldecanoyl-CoA 1S
Apparent Km = 680 ± 120 μM
Apparent Vmax = 301 ± 28 nmol/hr = 5.01 ± 0.46 nmol/min = 91.98 ± 8.45 nmol/min/mg
Apparent kcat = 72.48 ± 6.66 x 10-3 s-1
Apparent kcat/Km = 106.58 M-1 s-1
0.054 mg (1.15 nmoles) enzyme used per assay.
Michaelis-Menten
[Substrate] (μM)
0 200 400 600 800 1000 1200
Rat
e (n
mol
/hr)
0
50
100
150
200
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Lineweaver-Burk
1/[Substrate] (1/μM)
002 -0.001 0.000 0.001 0.002 0.003 0.004 0.005
1/R
ate
(hr/n
mol
)
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Direct Linear Plot
Apparent Km
-2000 -1000 0 1000 2000
App
aren
t Vm
ax
0
100
200
300
400
500
600
Figures based on whole data set
Apparent Km = 615 μM
Apparent Vmax = 290.9 nmol/hr = 4.84 nmol/min = 88.96 nmol/min/mg
Apparent kcat = 70.1 x 10-3 s-1
Apparent kcat/Km = 114 M-1 s-1
0.054 mg (1.15 nmoles) enzyme used per assay.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Residuals
[Substrate] (μM)
0 200 400 600 800 1000 1200
Res
idua
ls (n
mol
/hr)
-25
-20
-15
-10
-5
0
5
10
15
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Decanoyl-CoA 2, 325-650 μM substrate
Michaelis-Menten
[Substrate] (µM)
0 100 200 300 400 500 600 700
Rat
e (n
mol
/hr)
0
5
10
15
20
25
30
35
Apparent Km = 225 ± 415 μM
Apparent Vmax = 36.12 ± 21.45 nmol/hr = 0.601 ± 0.356 nmol/min = 10.63 ± 6.31 nmol/min/mg
Apparent kcat = 8.71 ± 5.17 x 10-3 s-1
Apparent kcat/Km = 38.71 M-1 s-1
0.054 mg (1.15 nmoles) enzyme used per assay.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Lineweaver-Burk
1/[Substrate] (1/µM)
006 -0.004 -0.002 0.000 0.002 0.004
1/R
ate
(hr/n
mol
)
0.02
0.04
0.06
0.08
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Direct Linear Plot
Apparent Km
000 -1500 -1000 -500 0 500 1000
App
aren
t Vm
ax
0
10
20
30
40
50
60
70
Plot above is for whole data set, 325-1625 μM
Estimates of Km and Vmax based on rates obtained at 325, 488 and 650 μM substrate only.
Apparent Km = 224 μM
Apparent Vmax = 34.78 nmol/hr = 0.579 nmol/min = 10.63 nmol/min/mg
Apparent kcat = 8.39 x 10-3 s-1
Apparent kcat/Km = 37.46 M-1 s-1
0.054 mg (1.15 nmoles) enzyme used per assay.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Residuals
[Substrate] (µM)
300 350 400 450 500 550 600 650 700
Res
idua
ls (n
mol
/hr)
-10
-8
-6
-4
-2
0
2
4
6
8
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010
Decanoyl-CoA, 325 – 1625 μM substrate showing non-competitive substrate inhibition.
Michaelis-Menten
[Substrate] (µM)
0 200 400 600 800 1000 1200 1400 1600 1800
Rat
e (n
mol
/hr)
0
5
10
15
20
25
30
35
Vmax = 12520 nmol/hr Km = 128400 μM Ki = 2 μM Equation used to fit data was for non-competitive substrate inhibition:
v = Vmax [S]/Km + [S] + [S]2/Ki
Where v = rate, Vmax = maximum rate, Km = Michaelis constant, [S] = substrate concentration, and
Ki = inhibition constant. Clearly the obtained values for kinetic parameters are unreasonable despite
graphical evidence for non-competitive substrate inhibition (above). Inspection of direct linear plot
shows deviation from Michaelis-Menten kinetics when considering all substrate concentrations.
Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2010